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Abstract

Fresnel Incoherent Correlation Holography (FINCH) enables holograms and 3D images to be created from incoherent light with just a camera and spatial light modulator (SLM). We previously described its application to microscopic incoherent fluorescence wherein one complex hologram contains all the 3D information in the microscope field, obviating the need for scanning or serial sectioning. We now report experiments which have led to the optimal optical, electro-optic, and computational conditions necessary to produce holograms which yield high quality 3D images from fluorescent microscopic specimens. An important improvement from our previous FINCH configurations capitalizes on the polarization sensitivity of the SLM so that the same SLM pixels which create the spherical wave simulating the microscope tube lens, also pass the plane waves from the infinity corrected microscope objective, so that interference between the two wave types at the camera creates a hologram. This advance dramatically improves the resolution of the FINCH system. Results from imaging a fluorescent USAF pattern and a pollen grain slide reveal resolution which approaches the Rayleigh limit by this simple method for 3D fluorescent microscopic imaging.

Figures (10)

Microscope configuration for holographic imaging. A fluorescent slide was positioned on the stage of the microscope and illuminated by standard epifluorescence methods. The illumination was controlled with a shutter to minimize photobleaching. The fluorescence emission passed through an input polarizer aligned with some angle to the polarization sensitive axis of the SLM. The emission beam reflected off of the SLM containing the appropriate diffractive lens patterns and then through an output polarizer before reaching the CCD camera.

Microscope configuration for SLM testing and alignment. A Coherent DPSS 532 nm or Thorlabs 633 nm laser passed through a Glan-Thompson polarizer and 20 × beam expander. The expanded laser beam was confirmed to be coherent and collimated with a shearing plate interferometer. The beam was directed to the microscope through a beam splitting cube mounted on the microscope turret which allowed the expanded laser beam to enter the microscope, reflect off the SLM and be directed to the camera or in some cases a power meter. Moving the turret to another position with a microscope objective made it possible to first obtain precision alignment of the microscope using the lasers and then to switch to imaging mode with objectives. The distance between the camera and SLM (zh) was varied by moving the camera along a precision track to confirm the focal lengths and characteristics of diffractive lens patterns.

Relationship between the radial parameter of the displayed quadratic phase patterns on the SLM at 532 nm and 633 nm and the distance of the measured plane of focus. Lines marked linear represent best fit lines calculated by the least squares method. The equations for the best fit lines are included.

Comparison of using a constant phase mask (a) versus the polarization method (b) to select and separate the plane and spherical waves in FINCH holography. Notice that when the polarization method is used, all the pixels on the SLM are used to create the diffractive lens pattern.

Best plane of focus reconstruction from holograms of the fluorescent USAF test slide using the constant phase mask technique and the polarizers method. (a) Static mask. (b) Input and output polarizers at 60 degrees. Olympus 20 × 0.75 NA objective. Bars labeled “a” are 1.6 microns thick and there is 2.5 microns distance between each of the three bars. Full bin 1 camera field of view, 300 microns square.

Best plane of focus from holograms of a pollen grain test slide using the constant phase mask technique and the dual polarizers method. (a) Constant phase mask. (b) Polarizers at 60 degrees. Olympus 20 × 0.75 NA objective. The full camera field of view of the microscopic image is 300 μm2.

Holograms of the USAF fluorescent test slide using an Olympus 20 × 0.75 NA objective. The input and output polarization orientation was changed as shown in the matrix and the phase 0° hologram from each series of three holograms (phase 0°,120° and 240°) is shown.

The best plane of focus from reconstructions of holograms of the USAF fluorescent test slide using an Olympus 20 × 0.75 NA objective is shown. The input and output polarization orientation varied as indicated.

Comparison of widefield and FINCH holographic imaging as a function of fluorescence emission bandwidth. The specimen was the USAF test pattern, imaged with an Olympus 20x 0.75 NA objective with a SLM-CCD distance of 400 mm. Columns I, II, and III respectively are widefield images, FINCH reconstructed images, and fluorescence emission spectra taken with varying emission filter combinations, as described in the text. Images and spectra in A, were taken with both a long pass and a standard emission bandpass filter, in B were taken with a standard emission bandpass filter and in C are were taken with only a long pass filter. The FWHM fluorescence emission (in nm) was ~17 nm for the narrow bandwidth (Row A), ~38 nm for the normal bandwidth (row B) and >50 nm bandwidth with a > 50 nm tail (Row C) for the wide bandwidth.emission fluorescence. The widefield images were obtained with input and output polarizers set at 0° with a 400 mm focal length diffractive lens pattern displayed on the SLM. The FINCH holograms were obtained with input and output polarizers set at 60° with an 800 mm focal length diffractive lens pattern displayed on the SLM. Best focus images were calculated from the holograms.